The present invention provides nucleotide sequences coding for a bacterial enolase enzyme. It encompasses processes for the fermentative preparation of amino acids using coryneform bacteria in which the eno gene is amplified.
L-amino acids, especially L-lysine, are used in the feeding of animals, in human medicine and in the pharmaceutical industry. They are typically produced by fermenting strains of coryneform bacteria, especially Corynebacterium glutamicum. Because of the great importance of amino acids, work is continually being done to improve production processes. Improvements may concern measures relating to the fermentation process (e.g., relating to stirring and oxygen supply) or the composition of the nutrient medium, (e.g., relating to the sugar concentration during the fermentation). They may also concern the purification of product (e.g., by ion-exchange chromatography) or the intrinsic performance properties of the microorganism itself.
To improve the performance properties of amino acid-producing microorganisms, methods of mutagenesis, selection and mutant selection are often employed. These methods may be used to obtain strains that are resistant to antimetabolites, such as, for example, the lysine analogue S-(2-aminoethyl)-cysteine, or which are auxotrophic for amino acids which are important in terms of regulation, and produce L-amino acids. In addition, methods of recombinant DNA technology have been used to improve the L-amino-acid-producing strains of Corynebacterium glutamicum by amplifying individual genes of amino acid biosynthesis. General articles on this subject include Kinoshita (xe2x80x9cGlutamic Acid Bacteria,xe2x80x9d in: Biology of Industrial Microorganisms, Demain and Solomon (eds.), Benjamin Cummings, London, UK, 1985, 115-142; Hilliger, BioTec 2:40-44 (1991); Eggeling, Amino Acids 6:261-272 (1994); Jetten, et al, Crit. Rev. Biotech. 15:73-103 (1995); and Sahm, et al., Ann. New York Acad. Sci. 782:25-39 (1996)).
The present invention is based upon the isolation of a bacterial gene coding for the enolase enzyme and the discovery that amino acid synthesis in coryneform bacteria is increased when the activity of this enzyme is enhanced. Bacteria may be treated in a number of different ways to enhance enolase activity but, most typically enhancement will result from bacteria being transformed with an expression vector encoding the enzyme. xe2x80x9cEnhancedxe2x80x9d refers to an increase in enolase activity in the treated bacteria relative to the amount of activity in untreated bacteria.
In its first aspect, the present invention is directed to an isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence coding for the eno gene. The polynucleotide is selected from the group consisting of: a) a polynucleotide that is at least 70% identical with a polynucleotide that codes for a polypeptide containing the amino-acid sequence of SEQ ID NO:2; and b) a polynucleotide that codes for a polypeptide containing an amino acid sequence that is at least 70% identical (and preferably 80 or 90% identical) with the amino-acid sequence of SEQ ID NO:2. In either case, the polypeptide must exhibit the activity of the enolase enzyme and, prefereably, it is a recombinant DNA replicative in coryneform bacteria.
The term xe2x80x9cisolatedxe2x80x9d means separated out of its natural environment. Thus, a protein or polypeptide that has been purified would be isolated for the purposes of the present invention. xe2x80x9cPolynucleotidexe2x80x9d in refers to polyribonucleotides and, preferably, polydeoxyribonucleotides, it being possible for these to be non-modified or modified RNA or DNA. xe2x80x9cPolypeptidesxe2x80x9d is understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.
A preferred polynucleotide coding for the enolase enzyme is that having the nucleotide sequence of SEQ ID NO:1. The invention includes not only polynucleotides having this specific sequence but also those that are at least 70% homologous (and preferably 80 or 90% homologous) to SEQ ID NO:1 and and in which one or more of the codons are replaced with a degenerate codon or in which there is a neutral sense mutation that does not alter the activity of the enolase enzyme. In addition, the invention encompasses a corynebacteria containing the any of the polynucleotides that are a part of the present invention.
Polynucleotide sequences derived from SEQ ID NO:1 may be used as hybridization probes for RNA, cDNA and DNA, in order to isolate the full length cDNA which coding for enolase and to isolate genes which have a high degree of homology with the enolase gene. Such oligonucleotides serving as probes or primers (e.g. for PCR) should be at least 15, and preferably at least 30 to 50 base pairs in length.
The inventors have found that improved activity occurs when the L-glutamic acid at position 223 of SEQ ID NO:2 is replaced by another proteinogenic amino acid. Thus, the invention includes enolase proteins with include this mutation and polypeptides which code for the mutated polypeptides. In a preferred embodiment, the L-glutamic acid at positon 223 is replaced with L-lysine as shown in SEQ ID NO:4. One way to code for a polypeptide to code for this is shown herein as SEQ ID NO:3 which is similar to SEQ ID NO:1 but with an adenine at position 817.
In another aspect, the invention is directed to a method for the production an L-amino acid, by: a) fermenting a coryneform bacteria that produces the amino acid and in which the activity of the enolase enzyme is enhanced; and b) isolating the L-amino acid from either the bacteria or from the medium used to grow the bacteria. Preferably, the amino acid being produced by the method is L-lysine and the enhancement of enolase activity results from the overexpression of the eno gene. The term xe2x80x9coverexpressionxe2x80x9d as used in this instance refers to an increase in the amount of mRNA transcribed relative to the amount of transcription occurring in untreated bacteria. The coryneform bacteria may be transformed with a nucleotide sequence coding for the enolase enzyme of SEQ ID NO:2 or in which the L-glutamic acid at position 223 of SEQ ID NO:2 is replaced by another proteinogenic L-amino acid, preferably L-lysine.
In addition to exhibiting enhanced enolase activity, the bacteria used to produce amino acids may have undergone additional genetic alterations to increase production. For example, the bacteria may be engineered to over-express one more genes encoding enzymes used in the biosynthetic pathway of of said L-amino acid or metabolic paths that reduce the formation of of the amino acid, e.g. L-lysine, may be at least partially eliminated. Specific genes that may be overexpressed to increase amino acid synthesis include: dapA gene coding for dihydrodipicolinate synthase; the lysC gene coding for a feedback-resistant aspartate kinase; the gap gene coding for glyceraldehyde-3-phosphate dehydrogenase; the tpi gene coding for triosephosphate isomerase; the pgk gene coding for 3-phosphoglycerate kinase; and the pyc gene coding for pyruvate carboxylase.